Hepatocellular carcinoma (HCC) accounts for 80-90% of primary liver tumors, and is one of the most common and devastating malignant diseases worldwide. Insulin-like growth factors (IGF-I and II) have been shown to play a key role in HCC by activating intracellular signaling cascades. Chief among these is the mitogen-activated protein kinase (MAPK) signaling pathway. Raf-1, the apical kinase, couples the MAPK pathway to extracellular tyrosine kinase receptors. Raf Kinase Inhibitory Protein (RKIP) is the prototype of a highly conserved family of proteins that bind directly to both Raf-1 and the next kinase in the pathway, MEK, disrupting their interaction, and antagonizing the activation of the entire signaling cascade. RKIP expression has been found to be reduced in breast, colon, liver and prostate cancers, among others. What is especially striking is the frequency of this event in HCC: almost 90% of human HCC specimens display reduced RKIP protein expression. Even more provocative are indications that RKIP may play a role in metastatic processes, implicating it as one of only a handful of known metastasis suppressor genes. This raises the possibility that modulation of RKIP protein expression or activity may have therapeutic value. These hopes are further fueled by observations that while RKIP protein expression is clearly reduced in many advanced stage tumors, it is seldom completely absent. The broad objective of this proposal is to initiate the development of mouse models to investigate in vivo the HCC tumor suppressor activity of RKIP. In pursuit of these goals two Specific Aims are proposed. The first will investigate the effect of downregulating RKIP. An already existing RKIP knockout will be used to determine the functional consequences of RKIP loss-of-function during the development and progression of HCC. The RKIP knockout will be combined with an exciting new mouse HCC model: a double transgenic with liver-specific expression of the hepatitis virus Bx transcriptional regulator (HBx) and insulin receptor substrate-1 (IRS-1). HBx/IRS-1 animals develop hepatocellular dysplasia that progresses to HCC. This model is of great interest because it recapitulates in the mouse many of the histological and molecular hallmarks of the human disease. The second aim will investigate the effect of restoring RKIP expression. A new transgenic model will be established to evaluate the functional consequences of upregulating RKIP during the development and progression of HCC. The approach will be to make a mouse with a liver-specific Tet- regulated RKIP transgene. These studies will provide critical in vivo functional evidence whether RKIP is a target with therapeutic potential. If affirmative, the in vivo data will justify future studies to unravel in detail the mechanisms that regulate RKIP expression and dysregulation in HCC as well as other cancers. PUBLIC HEALTH RELEVANCE: Hepatocellular carcinoma accounts for 80-90% of liver cancers, one of the most common malignancies worldwide. Intensive research has discovered the importance of a key cellular mechanism, the MAPK signaling pathway, in the development of this devastating disease. The main control point of the MAPK pathway, regulated by a protein kinase called Raf, has been targeted by the newly developed drug Nexavar, which has shown extraordinary promise in stage III clinical trials. Raf kinase is known to be influenced by other cellular regulators, Raf Kinase Inhibitory Protein (RKIP) being an especially important one. Epidemiological data has linked RKIP to cancer progression, raising the prospect that it may also be a valuable therapeutic target. The objective of this proposal is to develop mouse models of hepatocellular carcinoma in which this hypothesis can be directly tested.